Dendritic cells (DC) represent extremely potent antigen presenting cells and are the major cell type responsible for stimulating naive T cells. In this project we propose to use DC pulsed with carcinoembryonic antigen (CEA), alone or in combination with other antigens expressed by colorectal tumors, as a vaccine for the treatment of patients with advanced colorectal carcinoma. Our group in collaboration with Dr. Levy (Project 1) pioneered the use of antigen loaded DC in the treatment of cancer by demonstrating that such cells can induce anti-tumor responses in patients with malignant lymphoma, a relatively immunogenic tumor. However, DC immunotherapy is limited by the number of DC obtainable directly from blood, the inability of DC pulsed with unmodified protein to induce MHC class I restricted cytotoxic T lymphocytes (CTL), which are likely required to attach solid tumors, and the limited availability of well defined tumor antigens. To address the first of these problems we will use Flt3 ligand (FL), a hematopoietic growth factor, to mobilize DC precursors, in vivo. These cells will be harvested from blood, pulsed with CEA and infused as a vaccine. Preliminary results in ten patients treated with FL indicate that circulating DC increased by more than 20-fold and that the expanded cells, once harvested, activated, pulsed with a CEA peptide and infused as a vaccine, induced CTL and in some patients clinical responses. In the proposed project this trial will be expanded to a total of 30 patients. To address the problems of antigen access and processing we will evaluate several methods in model systems including exposure of DC to CEA protein or RNA in combination with immunostimulatory DNA (ISS), and transfer of whole tumor RNA or apoptotic bodies to DC. Exosomes derived from such DC will also be evaluated. Based on the results of these studies a pilot clinical trial will be undertaken that utilizes one or a combination of these strategies. By combining FL expansion of DC precursors with efficient methods of antigen transfer and DC activation, we hope to develop an effective, broadly applicable immunotherapeutic approach to colorectal cancer.